Transient Receptor Potential (TRP) channels are involved in many fundamental cell functions. Transient Receptor Potential Melastatin 2 (TRPM2), the second member of the TRPM subfamily to be cloned, is a widely expressed calcium-permeable channel activated by ADP-ribose (ADPR) and oxidative stress. A short physiological splice variant (TRPM2-S) inhibits Ca2+ influx through full length TRPM2 (L), functioning as a dominant negative. Preliminary data show that neuroblastoma xenografts expressing TRPM2-L have enhanced tumor growth and reduced sensitivity to doxorubicin compared to those expressing TRPM2-S, through modulation of hypoxia-inducible transcription factors (HIF-1/2?), mitochondrial function, and ROS. These fundamental observations form the basis of our Overall Hypothesis that TRPM2-L sustains cell proliferation and protects viability through moderate Ca2+ influx, which mediates expression of HIF-1/2?, maintains mitochondrial function, reduces ROS production and activates Pyk2. Understanding the function of TRPM2 channels in proliferation and survival and determination of the underlying mechanisms has high significance and clinical impact for many pathophysiological processes. Our aims are: Specific Aim 1: Is cell proliferation or viability reduced by inhibition of TRPM2 mediated Ca2+ influx? We will determine the role of TRPM2-mediated Ca2+ influx in modulation of cell viability, in vivo tumor formation, doxorubicin sensitivity, and ROS generation using TRPM2 loss and gain of function mutants. Specific Aim 2: Does TRPM2-mediated Ca2+ influx modulate mitochondrial function in neuroblastoma? Mitochondrial function including mitochondrial membrane potential, Ca2+ uptake, ATP production, and expression of BNIP3 and NDUFA4L2 are significantly reduced in cells expressing TRPM2-S, and ROS production is increased. This strongly supports the hypothesis that Ca2+ entry through TRPM2-L is important for normal mitochondrial function. (1) We will determine whether loss of Ca2+ influx in TRPM2 loss of function mutants inhibits mitochondrial function. (2) We will examine whether this is mediated through modulation of HIF-1?/2? and target mitochondrial proteins. (3) We will determine if TRPM2-L modulates mitophagy through BNIP3 expression. (4) We will examine the mechanism of regulation of ROS production by TRPM2. Specific Aim 3: Does TRPM2 modulate cell proliferation or viability through Ca2+-dependent Pyk2 activation? Our preliminary data show that proline-rich tyrosine kinase 2 (Pyk2), a Ca2+-sensitive, non-receptor tyrosine kinase, and its downstream targets ERK1/2 and CREB have reduced activation in TRPM2-S expressing cells. We will silence Pyk2, and use loss or gain of Pyk2 function mutants to assess the role of Pyk2 in TRPM2 modulation of proliferation, viability, CREB activation, and mitochondrial Ca2+ uptake. Understanding TRPM2 function has high significance and clinical impact because TRPM2 has an important role in cell survival after oxidative stress. Inhibition is a novel strategy to reduce tumor growth and enhance response to cancer therapeutics.